When hyperacute rejection of a discordant xenograft is prevented, grafts are still rejected within days, even in the absence of T cells or antibody, through a process known as delayed xenograft rejection (DXR). The apparently inexorable development of DXR despite any form of current immunosuppression, short of inducing gross immunodeficiency with concomitant high toxicity, is currently considered to be the main immunologic impediment to development of xenotransplantation as a clinical option. The syndrome of DXR is characterized by xenograft infiltration by cytokine-producing activated macrophages (MO) and natural killer (NK) cells, dense fibrin deposition and endothelial cell activation within the microvasculature. These features of DXR may be closely linked due to multiple interactions of the coagulation and inflammatory pathways. e.g. Activated protein C (aPC), protein S (PS) and thrombomodulin (TM), in addition to their known anticoagulant actions, constitute a physiologic pathway which modulates inflammatory addition to their known anticoagulant actions, constitute a physiologic pathway which modulates inflammatory events in vivo, though actions of this pathway are typically depressed during inflammation due to down-regulation of TM expression by endothelial cells by hypoxia or local production of cytokines such as TNF-alpha. Overall, this project will investigate the extent to which suppression of physiologic anticoagulant proteins is central to the immuno- pathogenesis of xenograft rejection, and whether enhancement of the function of aPC/PS/TM system may be of therapeutic significance in overcoming the development of DXR. To this end, the actions of aPC/PS/TM on each of the key cells (MO, NK and endothelial cells) involved in DXR will be determined, vis-a-vis cytokine production, induction of adhesion molecules and development of a procoagulant state. Next, we will investigate the membrane receptor(s) responsible for mediating the inhibitory effects of aPC on MO activation, using TNF-alpha production as a readout. In addition, since TM expression is a key requirement for efficient aPC generation, and given that MO express TM in a form which, in contrast to endothelial cells, is increased upon activation, the expression and regulation of TM on MO will be analyzed as a way to potentially boost aPC generation at sites of MO infiltration. Lastly, the effects of TM depletion or TM overexpression on inflammation and immunologic responses will be tested in vivo. In particular, by expressing TM constitutively or under control of a tetracycline-induced promoter, the biologic efficacy of heightened functioning of the aPC/TM pathway will be systematically evaluated in xenograft recipients.